Project Summary/Abstract The human immunodeficiency virus (HIV) is opposed by host cell factors, called ?restriction factors? with the potential to significantly control viral replication and affect disease progression and viral transmission. Our hypothesis is that the critical balance between the activity of restriction factors and the ability of the virus to antagonize or evade these factors plays in important role in HIV evolution, and ultimately, our ability to cure this infection. While some restriction factors are very active against HIV, others work poorly in humans or are polymorphic in the human population with both active and inactive versions. In the APOBEC3 locus of restriction factors, APOBEC3H stands out because some humans make active versions of this protein, while others do not, and a newly discovered polymorphism in APOBEC3C has increased activity in a subset of humans. Antagonism of all of the APOBEC3 proteins is dependent on the activity of the HIV-1 Vif protein which itself is polymorphic and must attack multiple host proteins at once. We will continue our studies on the interactions of restriction factors against HIV by determining the consequences of virus going from individuals with differing repertoires of APOBEC3 proteins. We will use an already established discordant couples cohort to understand the evolution and function of Vif proteins when virus is transmitted from an individual with one APOBEC3H genotype to a person with a different APOBEC3H genotype. In parallel, we will also exploit the natural infection of African Green Monkeys (AGMs) subspecies with divergent SIVs to understand how polymorphism in the APOBEC3 locus affects the evolution of the lentivirus-host relationship. In addition, we will determine the importance and mechanism of a gain-of-function polymorphism in APOBEC3C. We will furthermore create novel combinations of human APOBEC3 proteins in order to determine the limits of Vif evolution and potentially discover powerful antiviral proteins that have not been sampled by natural selection. Finally, we have initiated an innovative and flexible CRISPR/Cas9 screen for novel restriction factors that will provide further insights into the interactions between HIV and its host that affect virus replication. Overall, the goal of this proposal is to understand how the evolution and function of these restriction factors impacts HIV replication in humans.